Caps for sample wells and microcards for biological materials

ABSTRACT

A cover for a biological sample well tray, comprising a cap for sealing a sample well. The cap comprises a well lens for focusing light into the sample well and collecting light from the sample. In another aspect, the cap comprises an elongate portion configured to permit incoming light to pass into the sample well and out of the sample well. Various other aspects comprise a microcard for biological material, and an apparatus for a plurality of sample well strips. A method for testing a biological sample is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/099,644, filed Apr. 8, 2008, which is a division of U.S. applicationSer. No. 10/602,113, filed Jun. 23, 2003, which disclosures are hereinincorporated by reference in their entirety.

FIELD

The present teachings relate to devices and methods for containingsamples of biological material. The present teachings relate to welllenses for the samples of biological material that can be contained in asample container.

BACKGROUND

Biological testing has become an important tool in detecting andmonitoring diseases. In the biological testing field, polymerase chainreactions (PCR), ligase chain reactions, antibody binding reactions,oligonucleotide ligations assays, hybridization assays, and otherreactions can be used to analyze nucleic acids. In the biological field,cell-surface receptor binding assays, fluorescence-linked immunosorbentassays (FLISA), protein-protein interactions, enzyme assays, apoptosisassays, and other reactions can be used to analyze cells. Thesereactions have become valuable research tools with applications such ascloning, analysis of genetic expression, DNA sequencing, and drugdiscovery.

Recent developments in the field have led to an increased demand forbiological testing devices. Biological testing devices are now beingused in an increasing number of ways. It can be desirable to providereal-time detection capability in order to analyze on-going reactions.

In a real-time detection device, a plurality of lenses can be used tofocus light from a light source onto the samples to be tested, and tocollect the light emitted by the sample. These lenses can be bulky,taking up a considerable amount of space above the sample well tray. Theuse of a large number of components can make assembly and alignment ofthe optical components of the detection apparatus time consuming.Therefore, it can be desirable to have a simple, less complex structure.

SUMMARY

According to various embodiments, a cover for a biological sample welltray can comprise a cap for sealing a sample well. The cap can comprisesa well lens for focusing light into the sample well and substantiallycollimating light emitted by the sample. The well can comprise a welllens for focusing light into the sample well and collecting lightemitted by the sample. The well lens can be positioned in the side orbottom of the well.

According to various embodiments, a cover for a biological sample welltray can comprise a cap for sealing a sample well. The cap can comprisean elongate portion configured to permit incoming light to pass into thesample well and out of the sample well. The elongate portion cancomprise a well lens for focusing light into the sample well andcollecting light emitted by the sample. The elongate portion can bepositioned to provide a gap between cap and sample.

According to various embodiments, a microcard for biological material.can comprise a first member and a second member. The second member canprovide a plurality of sample chambers between the first member and thesecond member, the second member comprising a plurality of well lensescorresponding to the plurality of sample chambers. The well lenses canbe in fluid contact with a sample of biological material in the samplechamber. The well lenses can focus light into the sample well andcollecting light emitted by the sample.

According to various embodiments, an apparatus for a plurality of samplewell strips can be provided, wherein each sample well strip comprises aplurality of sample wells defined by side walls and bottoms, and aplurality of bottom stacking projections. Each bottom stackingprojection can extend downward from a sample well bottom. The bottomstacking projection can be configured to cap another sample well inanother sample well strip. The side walls can comprise a plurality ofwell lenses for focusing light into the sample well and collecting lightemitted by the sample.

According to various embodiments, a method for testing a biologicalsample comprises: providing a sample well or sample chamber containingsaid biological sample; providing a cap for the sample well, wherein thecap comprises a well lens; focusing light into the sample well; andcollecting light emitted by the sample.

It is to be understood that both the foregoing general description andthe following description of various embodiments are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments. Inthe drawings,

According to various embodiments, FIG. 1A illustrates a cross-sectionalview of a sample well of a sample well tray sealed with a transparentplastic sheet.

According to various embodiments, FIG. 1B illustrates a cross-sectionalview of a sample well of a sample well tray with a flat cap.

According to various embodiments, FIG. 1C illustrates a cross-sectionalview of a sample well of a sample well tray with a domed cap.

According to various embodiments, FIG. 1D illustrates a perspective viewof a sample well tray with a plurality of sample wells.

According to various embodiments, FIG. 2A illustrates a cross-sectionalview of a cap for a sample well, the cap comprising well lens.

According to various embodiments, FIG. 2B illustrates a cross-sectionalview of a cap for a sample well, the cap comprising a Fresnel well lens.

According to various embodiments, FIG. 3 illustrates a cross-sectionalview of a cap comprising a solid elongate portion and a separate welllens.

According to various embodiments, FIG. 4 illustrates a side view of thecap of FIG. 3.

According to various embodiments, FIG. 5 illustrates a cross-sectionalview of a cap comprising a solid elongate portion and a well lens.

According to various embodiments, FIG. 6A illustrates a cross-sectionalview of a cap comprising a hollow elongate portion positioned in asample well and a separate well lens.

According to various embodiments, FIG. 6B illustrates a cross-sectionalview of a cap comprising a hollow elongate portion similar to FIG. 6A.

According to various embodiments, FIG. 7 illustrates a side view of thecap comprising a hollow elongate portion of FIG. 6A.

According to various embodiments, FIG. 8 illustrates a cross-sectionalview of the cap comprising a hollow elongate portion of FIGS. 6A and 7.

According to various embodiments, FIG. 9A illustrates a cross-sectionalview of a cap comprising a hollow elongated portion and a well lens.

According to various embodiments, FIG. 9B illustrates a cross-sectionalview of a cap comprising a hollow elongate portion similar to FIG. 9A.

According to various embodiments, FIG. 10 illustrates a cross-sectionalview of a cap comprising a solid elongate portion and a well lens.

According to various embodiments, FIG. 11A illustrates a top view of amicrocard with a plurality of sample chambers comprising well lenses.

According to various embodiments, FIG. 11B illustrates a cross-sectionalview of the microcard of FIG. 11A, taken along line 11B-11B of FIG. 11A,showing the microcard positioned in a horizontal orientation.

According to various embodiments, FIG. 11C illustrates a cross-sectionalview of the microcard of FIG. 11A, showing the microcard positioned in avertical orientation.

According to various embodiments, FIG. 12 illustrates a cross-sectionalfrontal view of a plurality of sample well strips in a stackedconfiguration.

According to various embodiments, FIG. 13 illustrates a cross-sectionalside view of the plurality of sample well strips of FIG. 12.

According to various embodiments, FIG. 14 illustrates a top view of theplurality of sample well strips of FIG. 12.

According to various embodiments, FIG. 15A illustrates a cross-sectionalview of a sample well with a flat window on the bottom.

According to various embodiments, FIG. 15B illustrates a cross-sectionalview of a sample well with a well lens on the bottom.

According to various embodiments, FIG. 16 illustrates a cross-sectionalview of a sample well with a well lens on the side.

According to various embodiments, FIG. 17 illustrates a cross-sectionalview of a plurality of sample well strips in a stacked configuration,with well lenses on the bottom and side, and

According to various embodiments, FIG. 18 illustrates a cross-sectionalview of a sample well with an elongate portion leaving an air gap withsample.

According to various embodiments, FIG. 19 illustrates a cross-sectionalview of a cap comprising a hollow elongate portion similar to FIGS. 9Aand 9B, providing an air gap between the bottom surface of the lens anda sample.

DESCRIPTION OF VARIOUS EMBODIMENTS

Reference will now be made to various exemplary embodiments, examples ofwhich are illustrated in the accompanying drawings. Wherever possible,the same reference numbers are used in the drawings and the descriptionto refer to the same or like parts.

Although terms like “horizontal,” “vertical,” “upward,” “downward,”“side,” “front,” “upper,” and “lower” are used in describing variousaspects of the present teachings, it should be understood that suchterms are for purposes of more easily describing the teachings, and donot limit the scope of the teachings.

According to various embodiments, a sample well tray for biologicalsamples can be provided. An example of a sample well tray 10 with aplurality of sample wells 12 is shown in FIG. 1D. It should beunderstood that any type of sample well tray can be used. Sample welltrays typically have a plurality of sample wells for holding abiological sample. FIG. 1A shows a sample well 12 of a sample well tray10. A sample well tray can have a rectangular shape with a matrix ofsample wells 12 contained therein. Although FIG. 1D shows a sample welltray with 96 wells in a 8×12 matrix, it should be understood that thepresent invention is applicable for use with sample well trays havingany number of wells from one well to several thousand wells, such as,for example, 24 and 384 sample wells. Sample well trays having anynumber of sample well sizes can also be used. Although the term samplewell tray is used, it should be understood that many aspects of thepresent teachings are also suitable with other substrates such asmicrocard sample trays, where sample wells are replaced by samplechambers.

According to various embodiments, the sample well tray can be suitablefor incorporation into a number of different thermal cyclers, includingbut not limited to a 96-well Applied Biosystems thermal cycler. Thethermal cycler can be configured for thermally cycling samples ofbiological material. The thermal cycling device can be configured toperform nucleic acid amplification on samples of biological material.One common method of performing nucleic acid amplification of biologicalsamples is PCR. Various PCR methods are known in the art, as describedin, for example, U.S. Pat. Nos. 5,928,907 and 6,015,674 to Woudenberg etal., the complete disclosures of which are hereby incorporated byreference for any purpose. Other methods of nucleic acid amplificationinclude, for example, ligase chain reaction, oligonucleotide ligationsassay, and hybridization assay. These and other methods are described ingreater detail in U.S. Pat. Nos. 5,928,907 and 6,015,674.

According to various embodiments, the sample well tray can be used in athermal cycling device that performs real-time detection of the nucleicacid amplification of the samples in the sample disk during thermalcycling. Real-time detection systems are known in the art, as alsodescribed in greater detail in, for example, U.S. Pat. Nos. 5,928,907and 6,015,674 to Woudenberg et al., incorporated herein above. Duringreal-time detection, various characteristics of the samples are detectedduring the thermal cycling in a manner known in the art of nucleic acidamplification. Real-time detection can permit more accurate andefficient detection and monitoring of the samples during the nucleicacid amplification process. Alternatively, the sample well tray can beused in a thermal cycling device that performs endpoint detection of thenucleic acid amplification of the samples.

According to various embodiments, the sample well tray can be configuredto contact a sample block for thermally cycling the biological materialsin the sample wells of the sample well tray. The sample block can beoperatively connected to a temperature control unit programmed to raiseand lower the temperature of the sample block according to auser-defined profile. For example, in various embodiments, a user cansupply data defining time and temperature parameters of the desired PCRprotocol to a control computer that causes a central processing unit(CPU) of the temperature control unit to control thermal cycling of thesample block. Several non-limiting examples of suitable temperaturecontrol units for raising and lowering the temperature of a sample blockare described in U.S. Pat. No. 5,656,493 to Mullis et al. and U.S. Pat.No. 5,475,610 to Atwood et al., the disclosures of which are both herebyincorporated by reference for any purpose.

According to various embodiments, FIGS. 1A, 1B, and 1C show sealingstructures for sample wells in sample well trays where the well lensesare separate. For example, FIG. 1A is a cross-sectional view of a samplewell 12 of sample well tray 10 sealed with a transparent plastic sheetthat can be adhesive. The sample well 12 can include side walls 14 and abottom surface 16. The sample well 12 of FIG. 1A is configured to storea biological sample S in the interior of the side walls 14 and bottomsurface 16. According to various embodiments, the sample well can have aworking volume of approximately 200 μl. The volume of the sample wellscan vary anywhere from 0.1 μl to thousands of microliters (μl).According to various embodiments, a volume between 5 μl and 500 μl, or10 μl and 200 μl, or 50 μl can be used. In FIG. 1A, a thin, adhesive,transparent plastic sheet 18 is provided for sealing the top surface 20of the sample well 12. The sheet 18 can be made out of any suitablematerial such as hydrocarbon-based polymers compatible with PCR as knownin the art of nucleic acid amplification. These polymers can include,for example, polypropylene, polycarbonate, and polyethylene. Accordingto various embodiments, the sheet 18 is PCR-compatible. The sheet 18 canprovide an appropriate seal to the sample well 12.

According to various embodiments, as shown in FIG. 1A, a well lens 22can be provided adjacent to the sample well 12. The well lens 22 canfocus incoming light 24 from a light source (not shown) to a region inthe sample of biological material S. The well lens 22 can collect theemitted light from the sample of biological material S and direct ittoward the light detection apparatus (not shown). A wide variety of welllenses can be used for this purpose.

According to various embodiments, FIG. 1B shows a sample well tray 10with a flat cap 30 placed in the sample well 12. The flat cap 30includes a top portion 32 having a flat top surface 34 and a flat bottomsurface 36. Cap 30 further includes a cylindrical sealing structure 38.According to various embodiments, the flat bottom surface 36 forms anannular surface for engaging a top surface 20 of the sample well 12.Cylindrical sealing structure 38 extends downward into the sample well12 to engage the inside of side walls 14. The engagement of an outsidesurface 39 of the cylindrical sealing structure 38 serves to promotesealing of the sample well 12. The engagement of the flat bottom surface36 of the top portion of the cap 30 with the top surface 20 of thesample well 12 also serves to promote sealing of the sample well 12. Thesample well configuration of FIG. 1B further includes the well lens 22for directing the incoming light 24 into the sample S of biologicalmaterial, as discussed in greater detail with respect to FIG. 1A.

According to various embodiments, the cap 30 shown in FIG. 1B can beprovided individually, in strips, or in sheets in order to facilitatethe insertion and removal of the caps from the top of the sample wells12. In a 96-well configuration, the caps 30 can be provided in stripstypically having either 8 or 12 interconnected caps. In a 384-wellconfiguration, the caps can be provided in strips typically havingeither 16 or 24 interconnected caps.

According to various embodiments, FIG. 1C shows sample well tray 10 witha round domed cap 40 placed in the sample well 12. The round cap 40 issimilar to the flat cap 30 of FIG. 1B but has a slightly curved or domedcentral cap portion 42. The round cap 40 operates similarly to the flatcap. Both require the use of a separate well lens 22 positioned over thecap, for directing the incoming light 24 into the sample S of biologicalmaterial. The feature of a separate well lens takes up space above thesample well tray.

According to various embodiments, a cover for a biological sample welltray is provided. In one aspect, the cover comprises a cap for sealing asample well. The cap can comprise a well lens for focusing light intothe sample well from a light source and/or collect light emitted by thesample toward a detection apparatus.

According to various embodiments, such as illustrated in FIGS. 2A and2B, a cap with an integrated well lens is provided. As shown for examplein FIG. 2A, the sample well 12 can be provided with a cap 50 having awell lens 52 integrally formed into the cap 50. The well lens 52 canprovide focusing to the incoming light into the sample S of biologicalmaterial, without the need for a separate well lens. This can savespace, reducing costs and the number of parts and increases collectionefficiency. As shown in FIG. 2A, the well lens 52 can include a topsurface 54 and a bottom surface 58. The top surface 54 can be curved andthe bottom surface 58 can be flat. Incoming light 24 from a light source(not shown) passes through top surface 54 through to the bottom surface58 of the well lens 52 and into the sample well 12, focusing on a regionin the sample S of biological material. The well lens 52 can furtherserve to collect the light emitted from the biological material anddirect it toward the light detection apparatus (not shown). The term“emitted light” or grammatical variations thereof as used herein refersto the light emitted from the biological material that can includefluorescence emitted by the sample or dye molecules, for examplefluorescent dyes, upon excitation by the incoming light. Emitted lightcan also include light scattering off of objects in the biologicalsample. Emitted light can also include chemiluminescence,phosphorescence, and Raman scattering. As discussed previously, thelight detection can be performed either by an end-point detectionapparatus or a real-time detection apparatus, depending on the specificapplication.

According to various embodiments, the well lens can be of any of avariety of types known in the optics. FIG. 2A shows a cap comprising aplano-convex well lens. The well lens 52 can focus substantiallycollimated light on an object plane within the biological sample S andcollect light from an object plane in the biological sample andsubstantially collimate the light.

According to various embodiments, cap 50 and well lens 52 can be madeout of any acceptable material, such as polypropylene and otherhydrocarbon-based polymers. Other suitable materials include glass andacrylic. The material can be compatible with PCR as known in the art ofnucleic acid amplification. The cap and well lens can be manufactured byany known method, such as injection or compression molding, vacuumforming, and pressure forming. In the embodiment shown, the well lens 52is integrally formed with the cap 50. It should be understood howeverthat the well lens 52 could be attached by any known method.

According to various embodiments, the cap can include a cylindricalsealing member configured to engage an inner surface of the sample well.As shown in FIG. 2A, cap 50 can include a sealing structure 56integrally formed with the well lens 52 for promoting sealing of theinside of sample well 12. Sealing structure 56 is shown as a generallycylindrical member extending downward from the bottom surface 58 of thewell lens 52. An outer surface 59 of the cylindrical sealing structure56 can contact or otherwise engage the inner side wall of the samplewell 12 to assist in promoting sealing of the sample well from theoutside atmosphere. The outer surface 59 can be sized to have aninterference fit with the inner surface of the sample well 12.

According to various embodiments, the cap 50 can further include anouter annular flat bottom surface 60 for engaging the top surface 20 ofthe sample well 12. This engagement also serves to promote sealing ofthe sample well 12.

According to various embodiments, the caps of the present teachings aresuitable to fit in a wide variety of sample wells. FIG. 2A shows the cap50 in a sample well 12 with sidewalls 14 being tapered in a conicalmanner. It should be understood that the sidewalls 14 of the samplewells 12 can also be non-tapered, or any other suitable configuration.Additionally, although the bottom surface 16 is shown as being rounded,the bottom surface 16 can have any other suitable configuration, suchas, for example, flat. The cap can also be dimensioned to fit virtuallyany type of sample well. For example, with a 96-well sample well traysuch as shown in FIG. 1D, the caps will be designed and sized to fitwithin each sample well. With a 384-well sample tray, the size of thecaps will typically be smaller. It should be understood that thedimensions can greatly vary, depending on the size of the sample wells.Further, caps 50 can be manufactured into strips or sheets. The use ofstrips or sheets can facilitate the insertion and removal of the capsfrom the sample wells.

According to various embodiments, by integrating the well lens 52 intothe cap 50, the need for a separate well lens spaced from the samplewell 12 can be eliminated, thereby reducing the number of parts that areneeded. With fewer parts, concerns about alignment between the well lensand the sample well are reduced. Moreover, integrating several partsinto one can make the apparatus more compact, taking up less space.

According to various embodiments, the well lens can be a Fresnel lensintegrated into the sample well cap. FIG. 2B shows an embodiment whereinthe cap 70 includes a well lens 72 in the form of a Fresnel lens of thetype manufactured by Fresnel Technologies of Fort Worth, Tex. FIG. 2Bshows a Fresnel lens 72 integrated into a top surface 74 of the cap 70.The Fresnel lens 72 serves to focus the incoming light 24 from the lightsource (not shown) into the sample S of biological material. Fresnellenses are flat on one side and on the other side comprise a pluralityof ridges as shown in FIG. 2B. The angle of the ridges focuses the lighteven though the lens is flat. Among the characteristics of Fresnellenses is that they can be thin and easily be made of plastic. Fresnellenses can be used as a positive focal length collector or collimator.Fresnel lenses can have the power of convex lenses, but are thinner.This is an advantage because the optical path in the bulk of the lens issubstantially reduced. Bulk transmission losses are proportional tooptical path length, so reducing optical path length reducestransmission losses. The Fresnel lens 72 also serves to collect thelight emitted from the sample S to be tested. The use of the Fresnellens allows for optical detection without requiring a separate welllens.

According to various embodiments, the sample well cap is provided thatincludes an elongate member extending into the biological sample in thesample well. To the extent that any of the following structure issimilar to the structure described above, a detailed description willnot be repeated. For example, the sample well tray 10 and sample wells12 are assumed to be identical, or at least similar in importantaspects, to the sample well tray and sample well previously described.In the embodiments shown in FIGS. 3-10, a cap is provided for a samplewell of a sample well tray, the cap having an elongate portion.

According to various embodiments, the cap can be provided with anelongate member and a separate well lens. As shown for example in FIGS.3-4, a cap 110 can be provided for the sample well 12. The cap 110includes a top portion 112. In the embodiment shown in FIGS. 3-4, thetop portion 112 comprises a flat top surface 114 and an annular bottomsurface 130. The top portion 112 assists in sealing the inside of thesample wells 12 from the outside atmosphere. The top portion 112includes an annular bottom surface 130 for engaging a top surface 20 ofthe sample well 12 to promote sealing of the sample well 12, as shown inFIG. 3

According to various embodiments, the cap 110 can further comprise anintermediate portion 118 that extends from the top portion 112. In theembodiment shown, the intermediate portion 118 has a smaller diameterthan the top portion 112. The intermediate portion 118 is positionedbetween the top portion 112 and an elongate end portion 120 of the cap110.

In various embodiments, the intermediate portion 118 includes agenerally cylindrical sealing member 116 that can be sized so that anouter surface of the intermediate portion 118 engages the inner surfaceof the sample well side wall 14 upon insertion in the sample well 12.The intermediate portion 118 is shown as being solid in FIGS. 3-4. Theintermediate portion 118 shown in FIG. 3 includes an outer surface 122configured for mating with the inner surface of the side wall 14. Asshown in FIG. 3, the portion of the outer surface 122 that engages theinner surface of the side wall 14 has a greater diameter than theportion of the intermediate portion 118 that does not engage the sidewall 14. The outer surface 122 shown in FIG. 3 provides a line contactwith the sample well side wall 14 to assist in sealing the sample well12 from the outside atmosphere. It should be understood, thatalternatively, the outer surface 122 can have a constant diameter sothat a large amount of the outer surface engages the inner surface ofthe side wall 14. In alternative embodiments, a portion of the outersurface can include a separate ring or gasket to ensure a tight seal.The engagement assists in promoting a seal between the inside of thesample well 12 and the atmosphere.

According to various embodiments, the cap 110 can further include anelongate portion 120 extending from the intermediate portion 118 of thecap. Although shown as having a different diameter than the intermediateportion 118, it should be understood that the elongate portion 120could, in some embodiments, have the same diameter as the intermediateportion 118. Alternatively, the intermediate portion 118 can not berequired, so that the elongate portion can be all that is needed. In theembodiment shown in FIGS. 3-4, the elongate portion 120 is a solidcylinder with an outside diameter less than the diameter of theintermediate portion 118. It should be understood that the elongateportion 120 can be any other suitable shape. The elongate portion 120further includes a bottom surface 138 configured to contact the liquidsample S in the sample well.

According to various embodiments, the elongate portion 120 is configuredto permit incoming light to focus in a desired portion of the sample Sin the sample well 12. The incoming light can enter the cap 110 throughthe top surface 114 of the cap and then exit the cap 110 through thebottom surface 138 of the elongate portion 120, directly into the liquidsample. By being in direct contact with the liquid sample S in thesample well 12, condensation is prevented on the bottom surface 138 ofthe cap. It is desirable to avoid the formation of condensation on thebottom portion of a cap. Condensation can obstruct the optical path,thereby compromising the quality of the optical data. In variousembodiments air adjacent to the caps can permit condensation to occur onthe caps. In various embodiments, a heated cover raises the temperatureof the caps 110 and sample well 12 to the desired temperature to avoidcondensation. A heated cover, however, adds to the complexity of thedevice and increases costs. In various embodiments, the device can besimplified and costs can be reduced by providing the elongate portion120 of the cap 110 that directly contacts the sample S, as described inthe present teachings.

According to various embodiments, as shown in FIGS. 3-4, the cap 110,including the elongate portion 120, is solid. The cap 110 can be madeout of any material. According to various embodiments, the cap 110 ismade out of an optically clear PCR-compatible material. Examples ofsuitable materials include, but are not limited to, thermoplasticmaterials such as polypropylene, or polycarbonate resin marketed asLEXAN® by GE Plastics, Inc. (Pittsfield, Mass.) that provides hightransparency (light transmittance or clarity). Another suitable materialincludes resin-based polymethylpentene marketed as TPX® by MitsuiChemicals, Inc. (Tokyo, Japan) which provides high transparency andresistance to heat and chemicals. Other high transparency plastics whichcan be used for the cap include polymethyl methacrylate (PMMA) marketedas Lucryl® by BASF (Germany). The cap 110, including the elongateportion 120, can be formed by any known method, such as, for example,injection molding.

It should be understood that the cap 110 described in FIGS. 3-4 issuitable for use with any type of sample well. The sample well cap canbe suitably dimensioned for virtually any type of sample well and samplewell tray. In one example for a 96-well tray, the cap 110 shown in FIGS.3-4 has the following dimensions: top diameter (d2)=8.00 mm; elongateportion diameter (d1)=4.00 mm; and height (h1)=12.00 mm. Thesedimensions are for purposes of example only.

According to various embodiments, as shown in FIG. 3, a well lens can beprovided adjacent to the cap 110. The well lens 22 can be identical tothose described previously. The well lens 22 acts to direct incominglight from a light source (not shown) to a region in the sample ofbiological material S. The well lens 22 further serves to collect thelight emitted from the biological material and direct it toward thelight detection apparatus (not shown).

As discussed above, it is preferable to minimize the amount ofcondensation that occurs on the cap. According to various embodiments, aheater can be provided around the air gap A (see FIG. 3) above the toplevel of the liquid sample and below the intermediate portion 118. Invarious embodiments, a resistive, or other type of heater, could beprovided around the sample well side walls 14 in order to heat thesample well walls and prevent condensation from occurring on the insideof the sample well where air is located. In various embodiments, theheaters can be resistive heaters or Peltier heaters used to heat a metalsample block as known in the art of thermal cycling.

According to various embodiments, a cap can be provided with a well lensintegral with the cap, and an elongate portion extending into the samplewell. FIG. 5 shows a cap 140 similar to the cap shown in FIGS. 3-4, butwith a well lens 142 attached to or integral with the top portion 112 ofthe cap. The well lens 142 shown in FIG. 5 is a curved lens similar tothose described above. In the embodiment shown in FIG. 5, the well lens142 is integrated into the cap 140. The cap 140 can be formed in anyknown manner, such as injection or compression molding. The well lens142 is configured to direct incoming light to a desired position in theinterior of the sample well 12 and to direct reflected outgoing light toa detection apparatus (not shown).

According to various embodiments, by integrating the well lens 142 intothe cap 140, the need for a separate well lens spaced from the samplewell is eliminated, thereby reducing the number of parts that areneeded. This results in the features described regarding the embodimentillustrated in FIG. 2B, such as reducing concerns about alignment,making the apparatus more compact, and reducing costs.

According to various embodiments, a sample well cap with a hollowelongated portion can be provided. A hollow elongate portion can haveless bulk transmission loss than a solid elongate portion. As shown inFIGS. 6A, 7 and 8, a sample well cap 150 can be provided that has ahollow interior portion 152. The cap 150 includes a top portion 154 andan elongate portion 156. The top portion 154 includes an annular bottomsurface 158 for engaging a top portion 20 of the sample well 12 in amanner discussed earlier.

According to various embodiments, as shown for example in FIG. 6A, theelongate portion 156 of sample well cap 150 is defined by conical walls162 extending from the top portion 154 in a frusta-conical manner. Whenthe cap 150 is positioned on the sample well 12, the walls taper so thata space is provided between an outer surface 164 of the conical walls162 and an inner surface of the sample well side walls 14, except at thepoint of contact C between the conical walls 162 and the side walls 14.In the embodiment shown in FIG. 6A, the elongate portion 156 contactsthe side walls at an upper region thereof via notch 166. As shown inFIG. 6A, the diameter of the elongated portion 156 decreasesprogressively as the distance from the top portion 154 increases. Invarious embodiments, the progression of the elongated portion decreaseslinearly or exponentially.

According to various embodiments, the elongate portion 156 furtherincludes a bottom portion (or surface) 168. As shown in FIG. 6A forexample, the elongate portion 156 is configured so that the bottomsurface 170 of bottom portion 168 extends into and contacts the sampleof biological material S to be tested. Light can pass though bottomportion 168 for purposes of optical detection. With the embodimentillustrated in FIG. 6A, the incoming light 24 from the light source (notshown) passes through the well lens 22, through the hollow interiorregion of the cap, and then through the bottom portion 168 into thesample S to be tested. The reflected light can then pass back throughthe bottom portion 168 to the well lens 22 and to the detectionapparatus (not shown).

It should be understood that the cap 150 can be of any suitabledimension. In one example shown in FIG. 7, the cap has the followingdimensions: top portion diameter (d2)=8.00 mm; bottom surface diameter(d1)=4.00 mm; and height (h1)=12.00 mm. This embodiment reflects a96-well tray. The size of the cap is determined from the working volumeof the sample well, and will vary depending on the desired workingvolume. FIG. 8 shows a cross-section of cap 150.

According to various embodiments, as shown in FIG. 6B, the cap shown inFIG. 6A can have different dimensions and a different sample well.Sample well cap 150′ shown in FIG. 6B is similar to sample well cap 150shown in FIG. 6A. In FIG. 6B, the outer surface of the upper portion 174of the elongate portion 156′ is shown as having a smooth outer surface.In contrast, the outer surface of the upper portion of the elongateportion 156 shown in FIG. 6A has a notch 166.

According to various embodiments, a sample well cap is provided with ahollow elongate portion with a bottom surface incorporating a well lens.FIG. 9A illustrates a variation of the sample well cap shown in FIGS.6A, 7, and 8. As shown in FIG. 9A, the sample well cap 180 has a topportion and elongate portion 186 similar to that described in FIG. 6A,but further includes a well lens 182 integrally formed in a bottomsurface 184 of sample well cap 180. The well lenses in this embodimentare similar to those described above. The provision of a well lensintegral with the elongate portion eliminates the need for the separatewell lens positioned above the sample well cap. Eliminating the need fora separate well lens has all of the advantages previously discussed.

According to various embodiments, FIG. 9B shows a cap similar to thatshown in FIG. 9A, but with different dimensions, and a different samplewell. For most purposes, sample cap 180′ is similar to sample cap 180shown in FIG. 9A. According to various embodiments, as shown in FIG. 9B,the condensation cannot form on the bottom surface of the lens, becausethere is no air present, i.e. the bottom of the elongate portion 186 isimmersed in sample S. According to various embodiments, as shown in FIG.19, an air gap can be present between the bottom surface of the lens andsample S.

According to various embodiments, a sample well cap with an solidelongate portion and an integral well lens can be provided. FIG. 10shows a cap with a solid elongate tube, and a sealing member surroundingthe elongate tube. In the embodiment shown in FIG. 10, the cap 200includes a sealing member 202 and an elongate tube 204. The sealingmember 202 can be of any shape. In the embodiment shown in FIG. 10, thesealing member 202 is similar to the top and intermediate portions ofthe other embodiments. For example, sealing member 202 includes a bottomsurface 206 for contacting a top surface 20 of the sample well 12, andside surfaces 208 for contacting an inner surface of the sidewall of thesample well, to promote sealing in a manner discussed previously.Sealing member 202 can be constructed of a variety of suitablematerials, including hydrocarbon-based polymers compatible with PCR asknown in the art of nucleic acid amplification. The sealing member 202can include a through-hole 210 passing though the center thereof, forpermitting an elongate tube 204 to be fixed thereto. In the embodimentshown in FIG. 10, the elongate tube 204 is fixedly attached to thesealing member 202, in any known manner. The elongate tube 204 can alsobe integral with the sealing member 202.

According to various embodiments, as shown in FIG. 10, the bottom of theelongate tube 204 can include a curved lens 210. The curved lens 210serves to focus the incoming light from the light source (not shown)onto the sample S to be tested. The curved lens 210 is similar to thosepreviously discussed. In one embodiment, the curved lens 210 is aspherical convex lens.

According to various embodiments, a microcard for biological material isprovided. The microcard can comprise a first member, and a second memberdefining a plurality of sample chambers between the first member and thesecond member. In various embodiments, the second member can include aplurality of well lenses corresponding to the plurality of samplechambers. The well lenses are in fluid contact with a sample ofbiological material in the sample chamber. The well lenses focus lightinto the sample chamber and transmit light out of the sample chamber.

According to various embodiments, as shown in FIGS. 11A and 11B, asubstrate such as microcard 250 can be provided. A microcard is atwo-dimensional array of sample loci held within a continuous ornon-perforated substrate. The microcard can be flexible or rigid. Thesubstrate or microcard can contain any number of sample chambers forcontaining samples of biological material. The most typical number ofsample chambers is 60, 96, 384, or 1536, however, the microcard caninclude any number of sample chambers from one to at least severalthousand. FIG. 11A shows an example of a microcard 250 according to theembodiment of the present teachings.

According to various embodiments, as shown in FIG. 11B, the microcard250 includes a first member 262 and a second member 264. In theembodiment shown in FIG. 20B, the first member 262 includes all of thefeatures of the flow paths and sample chambers 266 in a polymeric sheet.A plurality of sample chambers 266 are defined between the first andsecond member.

According to various embodiments, the first member 262 can be made ofany suitable material such as a polymer. One such suitable polymer ispolypropylene. Other suitable polymers include, for example, polyester,polycarbonate, and polyethylene as described above. It can be desirableto make the first member 262 out of a PCR-compatible material. Thesecond member 264 is provided as a substantially flat plate that isattached to the first member 262 to complete the formation of thefeatures of the sample chambers and flow paths. The second member 264can be made out of any suitable material such as a metal foil.Alternatively, the second member could be made out of any of thepolymers suitable for use in the first member. The metal foil isparticularly suitable because it enhances the heat transfer to thesample chambers from a sample block (not shown) that is typicallypositioned under the microcard. The foil backing promotes the heating ofthe sample S to be tested to a desired temperature. The first and secondmembers are typically adhered to each other in order to create therequisite seal for the sample chambers.

According to various embodiments, as shown in FIG. 11B, the first member262 includes a plurality of raised portions 268 that each define a lens270 for light to pass through and focus on a region within the sample Sto be tested. The lenses 270 are similar to those described above. Inthe embodiment shown, the lens 270 includes a projection 274 thatextends downward into the sample to be tested. The projection is definedby an angled surface 276 and a flat bottom surface 278. The projectionis preferably sized so that the bottom surface contacts the sample to betested, as shown for example in FIG. 11B. According to variousembodiments, the condensation cannot form on the bottom surface of thelens, because there is no air present.

According to various embodiments, the microcard can be positioned on asample block for thermally cycling the biological material in the samplechambers. The sample block can be operatively connected to a temperaturecontrol unit programmed to raise and lower the temperature of the sampleblock according to a user defined profile. According to variousembodiments, the user can supply data defining time and temperatureparameters of the desired PCR protocol to a control computer that causesa central processing unit (CPU) of the temperature control unit tocontrol thermal cycling of the sample block. Several non-limitingexamples of suitable temperature control units for raising and loweringthe temperature of a sample block for a microcard or othersample-holding member or other sample-holding member are described inU.S. Pat. No. 5,656,493 to Mullis et al. and U.S. Pat. No. 5,475,610 toAtwood et al., incorporated herein above. Any suitable optical detectiondevice can also be used.

According to various embodiments, as shown in FIG. 11C, the microcard250 can be oriented vertically. The microcard 250 is identical to themicrocard shown in FIG. 11B. The lens 270 includes the projection thatextends into the sample chamber to contact the sample S to be tested.This reduces the possibility of condensation on the lens 270. As shownin FIG. 11C, in the case of a vertically oriented sample card, it ispreferable that the sample be of sufficient volume so that the flatsurface 278 of the projection 274 is immersed in liquid. No condensationcan form on the inside of the sample chamber at the flat surface 278because it is immersed in sample S.

According to various embodiments, an apparatus including a plurality ofsample well strips is provided. In various embodiments, a plurality ofsample wells are defined by side walls and bottoms. The sample wellstrips further comprise a plurality of bottom stacking projections, eachbottom stacking projection extending downward from a sample well bottom,the bottom stacking projection configured to cap another sample well inanother sample well strip. The side walls can comprise a plurality ofwell lenses for focusing light into the sample well and collecting lightemitted by the sample.

According to various embodiments, as shown in FIGS. 12-14, an apparatus300 with a plurality of sample well strips 310, 310′, and 310″ isprovided. FIG. 12 shows three rows of sample well strips (denoted as310, 310′, and 310″) stacked on top of each other, with a first row ofsample well strips 310 on the bottom, a second row of sample well strips310′ in the middle, and a third row of sample well strips 310″ on thetop thereof. For purposes of ease of discussion, the strips willgenerally be referred to by reference number 310, but will be referredto as 310, 310′, and 310″ when necessary. It should be understood thatalthough only three rows of sample well strips are shown in FIGS. 12-13,any suitable number of rows can be provided in accordance with thepresent teachings.

According to various embodiments, each of the sample well strips cancomprise a plurality of sample wells 320 defined by side walls 322 andbottom surfaces 324. The strips can contain any number of sample wells.In one embodiment, each strip includes 20 wells. Other sample wellstrips can include anywhere from two to several hundred wells. In oneembodiment, the sample wells 320 have a volume of approximately 5 μl,however, the size of the sample wells can vary from 0.001 μl tothousands of microliters (μl). In one embodiment, 48 sample well stripscan be stacked, each sample well strip containing 20 wells, for a totalof 960 wells. It should be understood that any number and size of samplewell strips can be envisioned with the present teachings.

According to various embodiments, the side walls 332 of the sample wells320 can be any suitable shape. According to various embodiments, asshown in FIGS. 12-14, the side walls are generally rectangular in shape,however, it is understood that the side walls can be any other shape,such as cylindrical. The side walls 322 define a top sample well opening348 that is generally rectangular. The top surface 330 of the side walls332 is generally flat in the example shown in FIGS. 12-14.

According to various embodiments, the sample well strips can furthercomprise a plurality of bottom stacking projections extending downwardfrom a sample well bottom. As shown in FIGS. 12 and 13, a bottomstacking projection 328 extends downward from each of the sample wellbottom surfaces 324. The bottom stacking projections 328 shown in FIGS.12 and 13 are shaped to be generally rectangular in order to mate with atop sample well opening 348 of an adjacent sample well strip. Accordingto various embodiments, there is a close interference fit between theouter surface 326 of the bottom stacking projections 328 and the innersurface 332 of the sample wells 320. According to various embodiments,the plurality of sample well strips have a snap-fit connection so thatthe sample well strips are securely fastened together. This close matingenhances the sealing inside of the sample wells 320. According tovarious embodiments, the open recess or well interior can be cylindricalto provide a maximum volume to surface area ratio.

According to various embodiments, as shown in FIGS. 12-14, the bottomstacking projection 328 includes an open recess 334. Open recess 334provides a head space such that side walls 322 are completely in fluidcontact with biological sample. The sample well bottom surfaces 324further define a bottom recess 344 around the bottom stackingprojections 328. The bottom recess 344 is a flat surface positionedbetween the bottom stacking projections 328. When the sample well strips310 are stacked, the top surface 330 of the side walls 322 engages theflat surface on the bottom recess 344. As discussed previously, thebottom stacking projections 328 can be sized so that there is aninterference fit in each of the sample wells when stacked. When stacked,the engagement of the top surface 330 of the side walls 322 with thebottom recess 344 further promotes sealing of the sample wells 320.

According to various embodiments, the sample well strips can include aplurality of lenses positioned in the side walls of the sample wellstrips. As shown in FIGS. 13 and 14, at least one lens 340 can bepositioned in the side wall of each sample well 320. In the embodimentshown in FIGS. 12-14, the lens is positioned on one of the front sidewalls, so that an optical detection device can be placed perpendicularto a front surface 342 of the plurality of lenses. Lenses 340 aresimilar to those described above.

According to various embodiments, the side of the sample well stripopposite the side with the lens will be referred to as the rear surface346, although it should be understood that terms such as “front” and“rear” are simply being used for purposes of more easily describing theteachings, and do not limit the scope of the teachings. The sample wellstrips include a rear surface 346. Since the optical detection isoccurring through the front surface 342, in some embodiments, a heatingmember (not shown) such as a sample block can be positioned against therear surface 346. This can assist in providing uniform temperatures foreach of the sample wells.

According to various embodiments, the sample well strips can be stackedvertically or horizontally. FIGS. 12-14 illustrate an embodiment inwhich the strips are stacked vertically, therefore the stackingoperation will be described for this embodiment. First, a predeterminedamount of sample to be tested is inserted into the sample wells of afirst sample well strip 310 by any known method, such as, for example,pipetting. Next, a second sample well strip 310′ is placed above thefirst sample well strip 310. The second sample well strip can already beloaded with sample, or can not be loaded with sample yet. The bottomstacking projections 328 of the second sample well strip 310′ are thenaligned with the top openings 348 of the sample wells of the firstsample well strip 310, and inserted therein. The bottom stackingprojections 328 of the second sample well strip 310′ should preferablysnap fit or have a close interference fit with the openings of thesample wells of the first sample well strip. If the second sample wellstrip has not been filled with liquid sample, it can be filled withsample at this time. This process can be repeated for all of theremaining strips. It should be noted that, in the embodiment shown inFIGS. 12-14, the top or third sample well strip 310′ will not typicallybe loaded with sample, but will instead be used as a cap for the secondsample well strip 310′. In alternative embodiments, the third samplewell strip 310″ can be loaded with sample.

According to various embodiments, the sample well strips can be stackedto provide excitation light from a light source that is substantiallyorthogonal from the light emitted by the sample. According to variousembodiments, as shown in FIG. 17, excitation light 24 a passes to thesample well strips 310 and 310′. Lenses 340 can collect emitted light 24b from the sample and transmit the collected light to a detector (notshown). Excitation light 24 a and emitted light 24 b can besubstantially orthogonal. According to various embodiments, sample wellstrips 310 and 310′ can comprise lenses to focus excitation light 24 ainto a region of the sample. Lenses 340 can collect emitted light 24 bfrom the sample and transmit the collected light to a detector.

According to various embodiments, the sample well tray can be used in adevice that performs fluorescence detection. Fluorescence detection isknown in the art for many applications including nucleic acids otherthan PCR, proteins, and cells. Fluorescent detection systems are knownin the art, as also described in greater detail in, for example, U.S.Pat. No. 6,130,745 to Manian et al., incorporated herein. According tovarious embodiments, fluorescence detection can provide high sensitivityto detect low levels of fluorescence. According to various embodiments,fluorescence detection can provide multiplexing to detect multipleexcitation and emission wavelengths in a sample.

According to various embodiments, the well lens can be positioned at thebottom of the sample wells to focus light coming into the sample wellfrom below the sample well tray and to collect light emitted by thesample. According to various embodiments, FIG. 15A illustrates a samplewell 12 comprising a flat window 410. Window 410 is a flat surface anddoes not focus the light 24 emitted from the sample. According tovarious embodiments, FIG. 15B illustrates a sample well 12 comprising awell lens 420 for collecting light 24 emitted from sample S. Well lens420 can focus excitation light 24 from a light source (not shown) into aregion of the sample S. Well lens 420 can be a plano-convex lens. Thelens radius can be chosen so that the center of curvature lies on theplano surface making the well lens aplanatic and increasing thenumerical aperture of the well lens by a factor related to the index ofrefraction of the well lens material without introducing substantialspherical aberration, coma, or astigmatism. According to variousembodiments, the apparatus can comprise an objective lens. The distancebetween the sample well tray and the objective lens can be decreased tocompensate for the well lens. According to various embodiments, theapparatus can comprise a beam expander. A beam expander can be used withthe well lens to provide substantially similar focus spot size of lightas the flat window. According to various embodiments, the sides 400 ofthe sample well can be constructed of a dark material to reduce emittedlight from escaping through sides 400. According to various embodiments,the well lens can be a Fresnel lens, or other lens known in the art ofoptics for focusing and collecting light. According to variousembodiments, as illustrated in FIG. 16, well lens 420 can be positionedat other locations on the sample well 12, such as sides 400.

According to various embodiments, the present teachings for sample wellsreceiving excitation light from above, sample well strips, and/or samplewells receiving excitation light from below can be combined. As shown inFIG. 18, sample well 12 can receive excitation light 24 a from a lightsource (not shown) through cap 70 comprising a Fresnel well lens 72integrated into the top surface 74 of cap 70. Well lens 72 can focusexcitation light 24 a into a region of sample S. Sample well 12 cancomprise concave well lens 420 on its side. Well lens 420 can collectemitted light 24 b from sample S in a direction substantially orthogonalto the direction of excitation light 24 a. According to variousembodiments, other combinations will be apparent to one skilled in theart.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure and methodsdescribed above. Thus, it should be understood that the presentteachings are not limited to the examples discussed in thespecification. Rather, the present teachings are intended to covermodifications and variations.

What is claimed is:
 1. A device for holding samples of biologicalmaterial, comprising: a sample well disposed about an axis, the samplewell comprising a topmost surface, a bottommost surface, and an interiorvolume configured to hold at least one sample of a biological material;and a cap disposed during use along the axis, the cap comprising anupper portion configured during use to be disposed above the topmostsurface of the sample well and an elongate lower portion configuredduring use to be disposed within the interior volume of the sample well,the lower portion comprising a bottommost surface; wherein during usethe bottommost surface of the lower portion is located closer to thebottommost surface of the sample well than to the topmost surface of thesample well; wherein the lower portion further comprises a conical walldefining a hollow interior volume; wherein the bottommost surface of thelower portion forms a bottom surface of the hollow interior volume;wherein the cap comprises a lens located at or near the bottommostsurface of the lower portion.
 2. The device of claim 1, furthercomprising: a plurality of sample wells disposed about respective axes,each sample well comprising a respective topmost surface, bottommostsurface, and interior volume; and a corresponding plurality of caps,each cap disposed during use along one of the respective axes, each capcomprising an upper portion configured during use to be disposed abovethe topmost surface of a respective sample well and an elongate lowerportion configured during use to be disposed within the interior volumeof the respective sample well.
 3. The device of claim 1, wherein thelower portion comprises a characteristic outer diameter and the capcomprises a characteristic length from a top most surface of upperportion to the bottommost surface of the lower portion, thecharacteristic length being at least twice the value of thecharacteristic outer diameter.
 4. A system for analyzing samples ofbiological material, comprising: a sample well disposed about an axis,the sample well comprising a topmost surface, a bottommost surface, andan interior volume configured to hold at least one sample of abiological material; a cap disposed along the axis, the cap comprisingan upper portion disposed above the topmost surface of the sample welland an elongate lower portion disposed within the interior volume of thesample well, the lower portion comprising a bottommost surface, thebottommost surface of the lower portion being located closer to thebottommost surface of the sample well than to the topmost surface of thesample well; wherein the lower portion further comprises a conical walldefining a hollow interior volume; wherein the bottommost surface of thelower portion forms a bottom surface of the hollow interior volume;wherein the cap comprises a lens located at or near the bottommostsurface of the lower portion.
 5. The system of claim 4, furthercomprising: a light source configured to provide excitation light to thesample well; and a detection system configured to receive fluorescentlight from the sample.
 6. The system of claim 4, further comprising: athermal cycler configured to configured to perform nucleic acidamplification on a sample of biological material.
 7. The system of claim4, further comprising: a plurality of sample wells disposed aboutrespective axes, each sample well comprising a respective topmostsurface, bottommost surface, and interior volume; and a correspondingplurality of caps, each cap disposed along one of the respective axes,each cap comprising an upper portion disposed above the topmost surfaceof the respective sample well and an elongate lower portion disposedwithin the interior volume of the respective sample well.
 8. The systemof claim 4, wherein the lower portion comprises a characteristic outerdiameter and the cap comprises a characteristic length from the upperportion to the bottommost surface of the cap, the characteristic lengthbeing at least twice the value of the diameter.